Markers of renal transplant rejection and renal damage

ABSTRACT

The present invention relates to methods of detecting renal transplant rejection and other forms of renal damage. Protein markers or renal damage are provided, along with assays for detecting said markers. Also provided are methods for identifying markers of renal damage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/850,231, filed Sep. 5, 2007 (now U.S. Pat. No. 8,021,895). The entiredisclosure of the aforesaid application is incorporated by reference inthe present application.

FIELD OF THE INVENTION

The present invention relates to methods of detecting renal transplantrejection and other forms of renal damage.

BACKGROUND TO THE INVENTION

Allograft rejection constitutes the major impediment to the success ofrenal transplantation. Chronic rejection is currently the most prevalentcause of renal transplant failure. Clinically, chronic rejectionpresents by chronic transplant dysfunction, characterized by a slow lossof function, often in combination with proteinuria and hypertension.Chronic rejection develops in grafts that undergo intermittent orpersistent damage from cellular and humoral responses resulting fromindirect recognition of alloantigens. The most effective option toprevent renal failure from chronic rejection is to avoid graft injuryfrom both immune and nonimmune mechanism together with nonnephrotoxicmaintenance immunosuppression. Currently available diagnostic methods,including clinical presentation and biochemical organ functionparameters, often fail to detect rejection until late stages ofprogression.

SUMMARY OF THE INVENTION

The inventors have identified biomarkers for detection of renalallograft rejection and other forms of renal damage. The inventionrelates to diagnosis and treatment of renal damage, including renaltransplant allograft rejection, using these biomarkers. Use of thebiomarkers includes use or detection of the proteins identified orfragments thereof, nucleic acids encoding said proteins or thecomplement thereof, and antibodies binding to said proteins.

Thus, the invention provides the use of the presence or amount of aprotein set out in Table 4 or Table 5 or a fragment thereof, orantibodies against said proteins, or nucleic acids encoding saidproteins or fragments thereof, as a marker for the diagnosis and/orprognosis of renal damage. Also provided is a method for the diagnosisand/or prognosis of renal damage, the method comprising determining thepresence or amount of a protein set out in Table 4 or Table 5, or afragment thereof, or antibodies against said proteins, or nucleic acidsencoding said proteins or fragments thereof in a sample from a patient.

Also provided is the use of a protein set out in Table 4 or Table 5 or afragment thereof, an antibody against one of said proteins, a nucleicacids encoding said proteins or a fragment thereof, or a nucleic acidwhich is the complement of a nucleic acids encoding said proteins or afragment thereof, in a method of diagnosis or prognosis of renal damage.

In some embodiments, prognosis and/or diagnosis is prognosis and/ordiagnosis of chronic rejection following renal transplantation, alsoknown as chronic allograft nephropathy (CAN). In other embodiments,prognosis and/or diagnosis may be prognosis and or diagnosis of renaldamage caused by disease or toxicity, for example

In one embodiment, the method comprises the steps of:

-   -   (a) contacting a sample from a patient with a solid support        having immobilised thereon a binding agent having binding sites        which are capable of specifically binding to the marker protein,        antibody or nucleic acid with a sample from a patient under        conditions in which the marker protein, antibody or nucleic acid        bind to the binding agent; and,    -   (b) determining the presence or amount of the marker protein,        antibody or nucleic acid bound to the binding agent.

Step (b) may comprise (i) contacting the solid support with a developingagent which is capable of binding to occupied binding sites, unoccupiedbinding sites or the marker protein, antibody or nucleic acid, thedeveloping agent comprising a label and (ii) detecting the label toobtain a value representative of the presence or amount of the markerprotein, antibody or nucleic acid in the sample.

The label may be, for example, a radioactive label, a fluorophor, aphosphor, a laser dye, a chromogenic dye, a macromolecular colloidalparticle, a latex bead which is coloured, magnetic or paramagnetic, anenzyme which catalyses a reaction producing a detectable result or thelabel is a tag.

In some embodiments, the binding agent is a protein set out in Table 4or Table 5, or a fragment thereof. In other embodiments, the bindingagent is an antibody which is capable of binding to a protein set out inTable 4 or Table 5, or a fragment thereof. In other embodiments, thebinding agent may be a nucleic acid which is complementary to thesequence of the nucleic acid to be detected.

In an alternative embodiment, the method comprises detecting a markerprotein or marker proteins in a sample from a patient using massspectroscopy. The proteins in the sample may be immobilised on a solidsupport. Optionally, the proteins may be subjected to proteolyticdigestion prior to analysis. Suitable analytical methods includeSELDI-TOF and MRM.

In some embodiments, the method comprises determining the presence oramount of a plurality of marker proteins, antibodies or nucleic acidsassociated with renal damage in a single sample. For example, aplurality of binding agents may be immobilised as predefined locationson the solid support.

Also provided is a kit for use in the diagnosis or prognosis of renaldamage by determining the presence or amount of an analyte selected froma protein set out in Table 4 or Table 5 or a fragment thereof, anantibody against said protein, and a nucleic acid encoding said proteinor a fragment thereof, in a sample from a patient, the kit comprising:

-   -   (a) a solid support having a binding agent capable of binding to        the analyte immobilised thereon;    -   (b) a developing agent comprising a label; and,    -   (c) one or more components selected from the group consisting of        washing solutions, diluents and buffers. The binding agent may        be as defined above. In particular, for detection of an        autoantibody, the binding agent may be a protein set out in        Table 4 or Table 5, or a fragment thereof. For detection of a        marker protein, the binding agent may be an antibody which is        capable of binding to a protein set out in Table 4 or Table 5,        or a fragment thereof. For detection of a nucleic acid, the        binding agent may be a nucleic acid which is complementary to        the sequence of the nucleic acid to be detected.

Also provided is a kit for use in the diagnosis or prognosis of renaldamage by determining the presence or amount of an analyte selected froma protein set out in Table 4 or Table 5, in a sample from a patient, thekit comprising one or more MRM peptides, as described below, in anassay-compatible format.

Also provided is the use of a protein set out in Table 4 or Table 5, ora fragment thereof, or antibodies capable of specifically binding theseproteins, for the preparation of a medicament for the treatment of renaldamage.

Further provided is the use of the presence or amount of a nucleic acidencoding any one of the proteins set out in Table 4 or Table 5 or afragment thereof as a marker for the diagnosis and/or prognosis of renaldamage.

In one embodiment, the invention provides a method for the diagnosisand/or prognosis of renal damage, the method comprising determining thepresence or amount of a nucleic acid encoding any one of the proteinsset out in Table 4 or Table 5, or a fragment thereof, in a sample from apatient.

Also provided is a kit for use in the diagnosis or prognosis of chronicrenal transplant rejection by determining the presence or amount of ananalyte selected from a protein set out in Table 4 or Table 5, or afragment thereof, or antibodies against these antigens, in a sample froma patient, the kit comprising:

-   -   (a) a solid support having a binding agent capable of binding to        the analyte immobilised thereon;    -   (b) a developing agent comprising a label; and,    -   (c) one or more components selected from the group consisting of        washing solutions, diluents and buffers.

Renal damage may include chronic rejection following renaltransplantation, also known as chronic allograft nephropathy, or renaldamage caused by disease or toxicity.

The methods of the invention are in most cases in vitro methods carriedout on a sample from a patient. The sample used in the methods describedherein may be a tissue or body fluid sample, for example a renal tissuesample, a blood or blood product (such as serum or plasma) sample or aurine sample. In a preferred embodiment, the sample is a depletedsample, i.e. a sample from which highly abundant proteins have beenremoved or depleted.

In preferred embodiments, said marker protein is any one ofN-acetylmuramoyl-L-alanine amidase precursor, adiponectin, AMBP proteinprecursor (α₁-microglobulin), C4b-binding protein α-chain precursor,ceruloplasmin precursor, complement C3 precursor, complement componentC9 precursor, complement factor D precursor, α_(1B)-glycoprotein,β₂-glycoprotein I precursor, heparin cofactor II precursor, Ig μ Chain Cregion protein, Leucine-rich α₂-glycoprotein precursor, pigmentepithelium-derived factor precursor, plasma retinol-binding proteinprecursor and translation initiation factor 3 subunit 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Analytical 2D-gel of serum sample C51-1 (comparison C1 vs. C3)

FIG. 2 Analytical 2D-gel of serum sample C51-1 (comparison C1 vs. C3)

FIG. 3 Analytical 2D-gel of serum sample D12-1 (comparison D1 vs. D3)

FIG. 4 Preparative 2D-gel of pooled control samples of study group

FIG. 5 Preparative 2D-gel of pooled disease samples of study group

FIG. 6 Histograms of the time course of protein regulations from disease(dark grey) and control (light grey) samples of table 4 at timepoints 1,2 and 3. Vertical axis: normalised spot volume. Horizontal axis:timepoints 1, 2 and 3.

A Spot 61 Plasminogen precursor

B Spot 210 Igμ chaim C region

C Spot 213 Igμ chaim C region

D Spot 214 Igμ chaim C region

E Spot 445 b2-glycoprotein I precursor

F Spot 453 Complement C1R subcomponent precursor

G Spot 461 b2 glycoprotein I precursor

H Spot 466 b2-glycoprotein I precursor

I Spot 553 Pigment epithelium-derived factor precursor

J Spot 557 Pigment epithelium-derived factor precursor

K Spot 559 Pigment epithelium-derived factor precursor

L Spot 563 Pigment epithelium-derived factor precursor

M Spot 570 Pigment epithelium-derived factor precursor

N Spot 673

O Spot 712 Transthyretin precursor

P Spot 722 a1-microglobulin

Q Spot 728 a1-microglobulin

R Spot 730 a1-microglobulin

S Spot 734 a1-microglobulin

T Spot 735 a1-microglobulin

U Spot 741 a1-microglobulin

Spot 760 Complement factor H-related protein 2 precursor

W Spot 771 Complement C3 precursor, a chain

X Spot 781 Complement C3 precursor, a chain/Complement C4 precursor

Y Spot 785 Complement factor D precursor

Z Spot 760 Complement D precursor

AA Spot 825 Plasma retinol-binding protein precursor

BB Spot 826 Plasma retinol-binding protein precursor

CC Spot 847 b2-glycoprotein precursor

FIG. 7 Histograms of the time course of protein regulations from disease(dark grey) and control (light grey) samples of table 4 at timepoints 1,2 and 3. Vertical axis: normalised spot volume. Horizontal axis:timepoints 1, 2 and 3.

A Spot 56 Ceruloplasmin precursor (Ferroxidase)

B Spot 113 Complement factor B precursor

C Spot 149 a2-macroglobulin precursor

D Spot 150 a2-macroglobulin precursor

E Spot 238 Inter-a-trypsin inhibitor heavy chain H1 precursor

F Spot 242 Inter-a-trypsin inhibitor heavy chain H1 precursor

Spot 243 Inter-a-trypsin inhibitor heavy chain H1 precursor

H Spot 463 Complement C1r subcomponent precursor

I Spot 464 Complement C1r subcomponent precursor

J Spot 498

K Spot 510 Hemopexin precursor (b1B-glycoprotein)

L Spot 515 Hemopexin precursor (b1B-glycoprotein)

M Spot 551 Hemopexin precursor (b1B-glycoprotein)

N Spot 562 Leucine-rich a2-glycoprotein precursor

O Spot 622 Apolipoprotein A-IV precursor

P Spot 657 Translation initiation factor 3 subunit 10/Ceruloplasminprecursor

Q Spot 711 Complement C4-pre/Complemen C4-b pre

R Spot 854 a1B-glycoprotein precursor

FIG. 8 PCA scores plot of the control data set containing all gels.

FIG. 9 PCA scores plot of the control data set without the outliersgels.

FIG. 10 PCA scores plot of the disease data set containing all gels.

FIG. 11 PLS-DA scores plot of the Control/1 and Control/3 groups.

FIG. 12 PLS-DA loading plots (spot numbers) of the Control_(—)1 andControl_(—)3 groups. In lighter type are indicated numbers of spotswhich have been selected through the 2D gel approach.

FIG. 13 PLS scores plots of the Disease/1 and Disease/3 groups.

FIG. 14 PLS-DA loading plots (spot numbers) of the Disease/1 andDisease/3 groups. In lighter type are indicated numbers of spots whichhave been selected through the 2D gel approach.

FIG. 15 PCA score plots of the control/1 and disease/1 groups.

FIG. 16 PLS scores plots of the sample/1 data set.

FIG. 17 PLS loadings plots of the sample/1 groups. In lighter type areindicated numbers of spots which have been selected through the 2D gelapproach.

FIG. 18 PCA score plots of the control/3 and disease/3 groups.

FIG. 19 PLS score plots of gels from the sample/3 data set.

FIG. 20 PLS loading plots from the sample/3 data set. In lighter typeare indicated numbers of spots selected through the 2D gel approach.

DETAILED DESCRIPTION Renal Damage

Renal damage may arise from, for example, transplant rejection, disease,toxicity and ischemic injury.

Renal transplant rejection is preferably chronic rejection, usuallydefined as rejection occurring six months or more after the transplant.Rejection may also be acute rejection, usually defined as rejectionoccurring up to six months after the transplant.

Disease may include Alport's Syndrome, Amyloidosis, Diabetes and thekidney, Fabry Disease, Focal and segmental Glomerulosclerosis (FSGS),Henoch-Schonlein purpura, Iga nephropathy (Berger's disease), InfantilePolycystic Disease, Kidney Stones, Haematuria Syndrome, Lupus and LupusKidney Disease, Membranoproliferative Glomerulonephritis (MPGN),Microscopic Polyarteris, Minimal Change Nepropathy, Reflux nephropathy,Renal artery stenosis, Vasculitis, Vesico-Ureteric Reflux and Wegenersgranulomatosis

Toxicity may be, for example, a side effect of drug treatment.

Marker Protein

The marker proteins disclosed herein may find use in diagnosis,prognosis or treatment in a variety of ways. The proteins themselves, orfragments thereof, may be detected in a diagnostic or prognostic assayas described below.

Marker proteins may be selected from any of the proteins shown in Table4 or Table 5. Preferred marker proteins are N-acetylmuramoyl-L-alanineamidase precursor, adiponectin, AMBP protein precursor(α₁-microglobulin), C4b-binding protein α-chain precursor, ceruloplasminprecursor, complement C3 precursor, complement component C9 precursor,complement factor D precursor, α_(1B)-glycoprotein, β₂-glycoprotein Iprecursor, heparin cofactor II precursor, Ig μ Chain C region protein,Leucine-rich α₂-glycoprotein precursor, pigment epithelium-derivedfactor precursor, plasma retinol-binding protein precursor andtranslation initiation factor 3 subunit 10.

The term “marker protein” or “biomarker” includes all biologicallyrelevant forms of the protein identified, including post-translationallymodified forms. For example, the marker protein can be present in thebody tissue in a glycosylated, phosphorylated, multimeric or precursorform.

Fragments of proteins to be detected or used for detection may be 10 ormore, 20 or more, 30 or more, 40 or more, 60 or more, 70 or more, 80 ormore, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more,350 or more, 400 or more or 500 or more amino acids in length.

Fragments of nucleic acids to be detected or used for detection may be10 or more, 20 or more, 30 or more, 40 or more, 60 or more, 70 or more,80 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 ormore, 350 or more, 400 or more, 500 or more, 600 or more, 700 or more,800 or more, 900 or more or 1000 or more basis in length.

Detection

A marker protein may have its expression modulated, i.e. quantitativelyincreased or decreased, in patients with renal damage. The degree towhich expression differs in normal versus damage states need only belarge enough to be visualised via standard characterisation techniques,such as silver staining of 2D-electrophoretic gels or immunologicaldetection methods including Western blotting or radioimmunoassay. Othersuch standard characterisation techniques by which expressiondifferences may be visualised are well known to those skilled in theart. These include successive chromatographic separations of fractionsand comparisons of the peaks, capillary electrophoresis, separationsusing micro-channel networks, including on a micro-chip, and massspectrometry methods including SELDI_TOF and MRM.

Chromatographic separations can be carried out by high performanceliquid chromatography as described in Pharmacia literature, thechromatogram being obtained in the form of a plot of absorbance of lightat 280 nm against time of separation. The material giving incompletelyresolved peaks is then re-chromatographed and so on.

Capillary electrophoresis is a technique described in many publications,for example in the literature “Total CE Solutions” supplied by Beckmanwith their P/ACE 5000 system. The technique depends on a applying anelectric potential across the sample contained in a small capillarytube. The tube has a charged surface, such as negatively chargedsilicate glass. Oppositely charged ions (in this instance, positiveions) are attracted to the surface and then migrate to the appropriateelectrode of the same polarity as the surface (in this instance, thecathode). In this electroosmotic flow (EOF) of the sample, the positiveions move fastest, followed by uncharged material and negatively chargedions. Thus, proteins are separated essentially according to charge onthem.

Micro-channel networks function somewhat like capillaries and can beformed by photoablation of a polymeric material. In this technique, a UVlaser is used to generate high energy light pulses that are fired inbursts onto polymers having suitable UV absorption characteristics, forexample polyethylene terephthalate or polycarbonate. The incidentphotons break chemical bonds with a confined space, leading to a rise ininternal pressure, mini-explosions and ejection of the ablated material,leaving behind voids which form micro-channels. The micro-channelmaterial achieves a separation based on EOF, as for capillaryelectrophoresis. It is adaptable to micro-chip form, each chip havingits own sample injector, separation column and electrochemical detector:see J. S. Rossier et al., 1999, Electrophoresis 20: pages 727-731.

Other methods include performing a binding assay for the marker protein.Any reasonably specific binding agent can be used. Preferably thebinding agent is labelled. Preferably the assay is an immunoassay,especially between the marker and an antibody that recognises theprotein, especially a labelled antibody. It can be an antibody raisedagainst part or all of the marker protein, for example a monoclonalantibody or a polyclonal anti-human antiserum of high specificity forthe marker protein.

Where the binding assay is an immunoassay, it may be carried out bymeasuring the extent of the protein/antibody interaction. Any knownmethod of immunoassay may be used. A sandwich assay is preferred. In anexemplary sandwich assay, a first antibody to the marker protein isbound to the solid phase such as a well of a plastics microtitre plate,and incubated with the sample and with a labelled second antibodyspecific to the protein to be assayed. Alternatively, an antibodycapture assay can be used. Here, the test sample is allowed to bind to asolid phase, and the anti-marker protein antibody is then added andallowed to bind. After washing away unbound material, the amount ofantibody bound to the solid phase is determined using a labelled secondantibody, anti- to the first.

In another embodiment, a competition assay is performed between thesample and a labelled marker protein or a peptide derived therefrom,these two antigens being in competition for a limited amount ofanti-marker protein antibody bound to a solid support. The labelledmarker protein or peptide thereof can be pre-incubated with the antibodyon the solid phase, whereby the marker protein in the sample displacespart of the marker protein or peptide thereof bound to the antibody.

In yet another embodiment, the two antigens are allowed to compete in asingle co-incubation with the antibody. After removal of unbound antigenfrom the support by washing, the amount of label attached to the supportis determined and the amount of protein in the sample is measured byreference to standard titration curves established previously.

The binding agent in the binding assay may be a labelled specificbinding agent, which may be an antibody or other specific binding agent.The binding agent will usually be labelled itself, but alternatively itmay be detected by a secondary reaction in which a signal is generated,e.g. from another labelled substance.

The label may be an enzyme. The substrate for the enzyme may be, forexample, colour-forming, fluorescent or chemiluminescent.

An amplified form of assay may be used, whereby an enhanced “signal” isproduced from a relatively low level of protein to be detected. Oneparticular form of amplified immunoassay is enhanced chemiluminescentassay. Conveniently, the antibody is labelled with horseradishperoxidase, which participates in a chemiluminescent reaction withluminol, a peroxide substrate and a compound which enhances theintensity and duration of the emitted light, typically 4-iodophenol or4-hydroxycinnamic acid.

Another form of amplified immunoassay is immuno-PCR. In this technique,the antibody is covalently linked to a molecule of arbitrary DNAcomprising PCR primers, whereby the DNA with the antibody attached to itis amplified by the polymerase chain reaction. See E. R. Hendrickson etal., Nucleic Acids Research 23: 522-529 (1995). The signal is read outas before.

The time required for the assay may be reduced by use of a rapidmicroparticle-enhanced turbidimetric immunoassay such as the typeembodied by M. Robers et al., “Development of a rapidmicroparticle-enhanced turbidimetric immunoassay for plasma fattyacid-binding protein, an early marker of acute myocardial infarction”,Clin. Chem. 1998; 44:1564-1567.

The full automation of any immunoassay contemplated in a widely usedclinical chemistry analyser such as the COBAS™ MIRA Plus system fromHoffmann-La Roche, described by M. Robers et al. supra, or the AxSYM™system from Abbott Laboratories, should be possible and applied forroutine clinical diagnosis of kidney damage including kidney transplantrejection.

Alternatively, the diagnostic sample can be subjected to two dimensionalgel electrophoresis to yield a stained gel and the increased ordecreased concentration of the protein detected by an increased ordecreased intensity of a protein-containing spot on the stained gel,compared with a corresponding control or comparative gel. The inventionincludes such a method, independently of the marker proteinidentification given above.

In another method, the diagnostic sample can be subjected toSurface-Enhanced Laser Desorption Ionisation—Time of Flight massspectrometry (SELDI-TOF). In this method the sample is typically a bodyfluid and is added to the surface of a SELDI-TOF ProteinChip prior toanalysis in the SELDI-TOF mass spectrometer. General methods ofSELDI-TOF analysis for human tissue samples are provided ininternational patent application WO 01/25791. The ProteinChip systemconsists of aluminium chips to which protein samples can be selectivelybound on the surface chemistry of the chip (e.g. anionic, cationic,hydrophobic, hydrophilic etc). Bound proteins are then co-crystallisedwith a molar excess of small energy-absorbing molecules. The chip isthen analysed by short intense pulses of N2 320 nm UV laser with proteinseparation and detection being by time of flight mass spectrometry.Spectral profiles of each group within an experiment are compared andany peaks of interest can be further analysed using techniques asdescribed below to establish the identity of the protein.

The diagnostic sample can be subjected to analysis by multiple reactionmonitoring (MRM) on an ion-trap mass spectrometer. Based on the massspectrometry profiles of the marker proteins described below singletryptic peptides with specific known mass and amino acid sequences areidentified that possess good ionising characteristics. The massspectrometer is then programmed to specifically survey for peptides ofthe specific mass and sequence and report their relative signalintensity. Using MRM it is possible to survey for up to 5, 10, 15, 20,25, 30, 40, 50 or 100 different marker proteins in a single LC-MS run.The intensities of the MRM peptides of the marker proteins in thediagnostic sample are compared with those found in samples from subjectswithout kidney damage and/or kidney transplant rejection allowing thediagnosis of kidney damage and/or kidney transplant rejection to bemade.

The MRM assay can be made more truly quantitative by the use of internalreference standards consisting of synthetic absolute quantification(AQUA) peptides corresponding to the MRM peptide of the marker proteinwherein one or more atoms have been substituted with a stable isotopesuch as carbon-14 or nitrogen-15 and wherein such substitutions causethe AQUA peptide to have a defined mass difference to the native MRMpeptide derived from the diagnostic sample. By comparing the relativeion intensity of the native MRM and AQUA peptides the true concentrationof the parent protein in the diagnostic sample can thus be determined.General methods of absolute quantitation are provided in Gerber, ScottA, et al. “Absolute quantification of proteins and phosphoproteins fromcell lysates by tandem MS” PNAS, Jun. 10, 2003. Vol 100. No 12. p6940-6945.

Alternatively, antibodies to marker proteins may be detected in apatient sample, using marker proteins or fragments thereof as adetection agent, for example in an ELISA. An altered concentration ofmarker proteins may be detected by detecting the presence or an alteredamount of autoantibody thereto, compared with the level of autoantibodyin a control sample. The level of autoantibody can be detected byWestern blot (from 1D or 2D electrophoresis) against kidney tissueobtained from biopsy or surgical removal, or kidney-derived cell linesgrown in vitro, or by enzyme-linked immunosorbent assay (ELISA), proteinmicroarray or bead suspension array using purified proteins.

By way of example, detection of autoantibodies to proteinsdifferentially increased in kidney damage and/or kidney rejectionpatients can be carried out as follows. Recombinant proteins areexpressed in baculovirus infected insect cells and used to coat thesurface of microtitre plates. A sample from patients suspected of havingkidney damage and/or kidney rejection is added to duplicate wells ofeach microtitre plate and incubated at 37° C. for 1 hour. Plates areaspirated and washed prior to the addition of a horse-radish peroxidase(HRP) labelled anti-human IgG antiserum and incubated for 1 hour at 37°C. Finally, binding of the antihuman antiserum is revealed by aspiratingthe plates, washing, and then adding tetra-methylbenzidine (TMB) whichin the presence of HRP produces a coloured product the intensity ofwhich is measured by reading the plates at 450 nm. An identical set ofplates is tested with the exception that the second antibody is a HRPlabelled anti-human IgM antiserum. The levels of IgG and/or IgMautoantibodies to each of the kidney damage and/or kidney rejectionmarker proteins is altered when compared to the levels found in samplesfrom healthy individuals.

It is contemplated within the invention to use (i) an antibody chip orarray of chips, or a bead suspension array capable of detecting one ormore proteins that interact with that antibody; or (ii) a protein chipor array of chips, or bead suspension array capable of detecting one ormore autoantibodies that interact with the marker proteins; or (iii) acombination of both antibody arrays and protein arrays.

An “antibody array” or “antibody microarray” is an array of uniqueaddressable elements on a continuous solid surface whereby at eachunique addressable element an antibody with defined specificity for anantigen is immobilised in a manner allowing its subsequent capture ofthe target antigen and subsequent detection of the extent of suchbinding. Each unique addressable element is spaced from all other uniqueaddressable elements on the solid surface so that the binding anddetection of specific antigens does not interfere with any adjacent suchunique addressable element.

A “bead suspension array” is an aqueous suspension of one or moreidentifiably distinct particles whereby each particle contains codingfeatures relating to its size and colour or fluorescent signature and towhich all of the beads of a particular combination of such codingfeatures is coated with an antibody with a defined specificity for anantigen in a manner allowing its subsequent capture of the targetantigen and subsequent detection of the extent of such binding. Examplesof such arrays can be found at www.luminexcorp.com where application ofthe xMAP® bead suspension array on the Luminex® 100™ System isdescribed.

Alternatively, differential expression of nucleic acids encoding markerproteins may be used as a detection method.

Expression of nucleic acid may be detected by methods known in the art,such as RT-PCR, Northern blotting or in situ hybridisation such as FISH.

The diagnosis can be based on the differential expression of one, two,three or more of the marker proteins, or of one, two, three or moreautoantibodies raised against such proteins, or a combination of both.Further, it can be part of a wider diagnosis in which two or moredifferent diseases are diagnosed. Renal damage and kidney transplantrejection can be diagnosed together with at least one other disease,which may or may not be a renal disease, in the same sample of bodytissue, by a method which includes detecting a change in concentrationof another protein in the diagnostic sample, compared with a sample of acontrol, normal human subject. These other disease(s) can be any whichare diagnosable in a body tissue.

Diagnosis and Prognosis

The term “diagnosis”, as used herein, includes the provision of anyinformation concerning the existence, non-existence or probability ofrenal damage, disease or renal transplant rejection in a patient. Itfurther includes the provision of information concerning the type orclassification of the disorder or of symptoms which are or may beexperienced in connection with it. It encompasses prognosis of themedical course of the condition.

The methods described herein are useful for both the diagnosis and/orprognosis of renal damage, including chronic renal transplant rejection.Renal damage or rejection may be indicated if one or more markers ispresent at increased or decreased concentration. For some markers, bothan increased or a decreased concentration may be indicative of damage orrejection.

The invention can be used to determine the stage of progression ofkidney damage including kidney transplant rejection, if desired, withreference to results obtained earlier from the same patient or byreference to standard values that are considered typical of the stage ofthe disease. In this way, the invention can be used to determinewhether, for example after treatment of the patient with a drug orcandidate drug, the disease has progressed or not. The result can leadto a prognosis of the outcome of the disease.

The methods typically employ a biological sample from patient such asblood, serum, tissue, plasma, urine or other suitable body fluids. Apreferred patient sample is blood or blood products such as serum orplasma. Plasma or other sample fluids may be depleted of high abundanceproteins prior to testing, as described below. Alternatively, thediagnostic sample may be a tissue section which is fixed, e.g. byfreezing or embedding in paraffin.

The diagnostic methods and uses of the invention may comprise the stepsof (i) detecting marker protein, nucleic acid or antibody expression ina test sample or samples, (ii) detecting marker protein, nucleic acid orantibody expression in a control sample or samples, and (iii) comparingthe level of expression on the control and test samples to establishwhether the marker is differentially expressed in the test sample.Alternatively, a pre-existing control may be used for comparison.Preferably, the control sample is from the same tissue or body fluidtype as the test sample.

In general, the term “control” refers to a normal human subject, i.e.one not suffering from renal damage, or to healthy tissue of the samehuman subject as the diagnostic sample. In some cases, particularly whenthe stages of renal transplant rejection are being monitoredprogressively, or when a course of treatment is being monitored, acomparison may instead be made with the concentration previously seen inthe same subject at an earlier stage of progression of the disease, orat an earlier time point.

The step of comparing with a control sample may not always be necessary,since in many cases it will be obvious to the skilled practitioner thatthe concentration is abnormally high or low.

“Differential expression”, as used above, refers to at least onerecognisable difference in protein or nucleic acid expression. It may bea quantitatively measurable, semi-quantitatively estimatable orqualitatively detectable difference in tissue or body fluid pexpression. Thus, a differentially expressed protein or nucleic acid maybe strongly expressed in tissue or body fluid in the normal state andless strongly expressed or not expressed at all in the damaged state.Conversely, it may be strongly expressed in tissue or body fluid in thedamage state and less strongly expressed or not expressed at all in thenormal state. Further, expression may be regarded as differential if theprotein or nucleic acid undergoes any recognisable change between thetwo states under comparison.

Samples may be taken daily, weekly, monthly or bimonthly aftertransplant. Daily or weekly samples may be appropriate for detection ofacute rejection, while monthly or bimonthly samples are more appropriatefor detection of chromic rejection.

Treatment

It will be understood that where treatment is concerned, treatmentincludes any measure taken by the physician to alleviate the effect ofrenal damage or transplant rejection on a patient. Thus, althoughreversal of the damage or elimination of the damage or effects ofrejection is a desirable goal, effective treatment will also include anymeasures capable of achieving reduction in the degree of damage orseverity of the effects.

In one aspect, the invention provides a method of treatment by the useof an agent that will restore the expression of one or moredifferentially expressed proteins in the kidney damage and/or kidneytransplant rejection state towards that found in the normal state inorder to prevent the development or progression of kidney damage and/ororgan transplant rejection. Preferably, the expression of the protein isrestored to that of the normal state.

In a further aspect, the present invention provides a method whereby thepattern of differentially expressed proteins in a sample from anindividual with kidney damage and/or kidney transplant rejection is usedto predict the most appropriate and effective therapy to alleviate thekidney damage and/or kidney transplant rejection

Assay Methods

Also provided is a method of screening an agent to determine itsusefulness in treating a kidney damage and/or kidney transplantrejection, the method comprising:

(a) obtaining a sample of from, or representative of, a subject havingkidney damage and/or kidney transplant rejection symptoms, who or whichhas been treated with the agent being screened;

(b) determining the presence, absence or degree of expression of amarker protein or proteins as disclosed herein in the sample from, orrepresentative of, the treated subject; and,

(c) selecting or rejecting the agent according to the extent to which itchanges the expression, activity or amount of the marker protein orproteins in the treated subject having kidney damage and/or kidneytransplant rejection symptoms.

Preferably, the agent is selected if it converts the expression of thedifferentially expressed protein towards that of a normal subject. Morepreferably, the agent is selected if it converts the expression of theprotein or proteins to that of the normal subject.

Also provided is a method of screening an agent to determine itsusefulness in treating kidney damage and/or kidney transplant rejection,the method comprising:

(a) obtaining over time samples from, or representative of, a subjecthaving kidney damage and/or kidney transplant rejection symptoms, who orwhich has been treated with the agent being screened;

(b) determining the presence, absence or degree of expression of amarker protein or proteins as disclosed herein in said samples; and,

(c) determining whether the agent affects the change over time in theexpression of the marker protein in the treated subject having kidneydamage and/or kidney transplant rejection symptoms.

Samples taken over time may be taken at intervals of weeks, months oryears. For example, samples may be taken at monthly, two-monthly,three-monthly, four-monthly, six-monthly, eight-monthly ortwelve-monthly intervals.

A change in expression over time may be an increase or decrease inexpression, compared to the initial level of expression in samples fromthe subject and/or compared to the level of expression in samples fromnormal subjects. The agent is selected if it slows or stops the changeof expression over time.

In the screening methods described above, subjects having differentiallevels of protein expression comprise:

(a) normal subjects and subjects having kidney damage and/or kidneytransplant rejection symptoms; and,

(b) subjects having kidney damage and/or kidney transplant rejectionsymptoms which have not been treated with the agent and subjects havingkidney damage and/or kidney transplant rejection symptoms which havebeen treated with the agent.

Compositions

The markers or antibodies thereto described herein may be formulatedinto compositions, in particular therapeutic compositions. Thepreparation of therapeutic compositions which contain polypeptides orproteins as active ingredients is well understood in the art.Therapeutic compositions may be liquid solutions or suspensions, solidforms suitable for solution in, or suspension in a liquid prior toingestion may also be prepared. The therapeutic may also be emulsified.The active therapeutic ingredient is typically mixed with inorganicand/or organic carriers which are pharmaceutically acceptable andcompatible with the active ingredient. The carriers are typicallyphysiologically acceptable excipients comprising more or less inertsubstances when added to the therapeutic composition to confer suitableconsistencies and form to the composition. Suitable carriers are forexample, water, saline, dextrose, glycerol, and the like andcombinations thereof. In addition, if desired the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents and pH buffering agents which enhance theeffectiveness of the active ingredient. Therapeutic compositionscontaining carriers that have nutritional value are also contemplated.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc, a fusion protein of theinvention optional pharmaceutical adjuvants in a carrier, such as, forexample, water, saline aqueous dextrose, glycerol, ethanol, and thelike, to thereby form a solution or suspension. If desired, thecomposition to be administered may also auxiliary substances such as pHbuffering agents and the like. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, 20th Edition, 2000,pub. Lippincott, Williams & Wilkins.

Administration

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration. For oral administration, it has beenfound that oligonucleotides with at least one 2′-substitutedribonucleotide are particularly useful because of their absorption anddistribution characteristics. U.S. Pat. No. 5,591,721 issued to Agrawalet al. Oligonucleotides with at least one 2′-O-methoxyethyl modificationare believed to be particularly useful for oral administration.

Where a composition as described herein is to be administered to anindividual, administration is preferably in a “prophylacticallyeffective amount” or a “therapeutically effective amount” (as the casemay be, although prophylaxis may be considered therapy), this beingsufficient to show benefit to the individual.

Administration may be, for example, daily, weekly or monthly. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of what is being treated. It will alsodepend upon toxicity of the therapeutic agent, as determined bypre-clinical and clinical trials. Prescription of treatment, e.g.decisions on dosage etc, is within the responsibility of generalpractitioners and other medical doctors.

Dosing

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a reduction in viral titre (routinely measuredby Western blot, ELISA, RT-PCR, or RNA (Northern) blot, for example) iseffected or a diminution of disease state is achieved. Optimal dosingschedules are easily calculated from measurements of drug accumulationin the body. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Therapeutically orprophylactically effective amounts (dosages) may vary depending on therelative potency of individual compositions, and can generally beroutinely calculated based on molecular weight and EC50s in in vitroand/or animal studies. For example, given the molecular weight of drugcompound (derived from oligonucleotide sequence and chemical structure)and an experimentally derived effective dose such as an IC.sub.50, forexample, a dose in mg/kg is routinely calculated. In general, dosage isfrom 0.001 .mu.g to 100 g and may be administered once or several timesdaily, weekly, monthly or yearly, or even every 2 to 20 years.

Nucleic Acids

Nucleic acid may of course be double- or single-stranded, cDNA orgenomic DNA, or RNA. The nucleic acid may be wholly or partiallysynthetic, depending on design. Naturally, the skilled person willunderstand that where the nucleic acid according to the inventionincludes RNA, reference to the sequence shown should be construed asreference to the RNA equivalent, with U substituted for T. The presentinvention also encompasses the expression product of any of the nucleicacid sequences disclosed and methods of making the expression product byexpression from encoding nucleic acid therefore under suitableconditions in suitable host cells.

Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Current Protocols inMolecular Biology, Ausubel et al. eds., Wiley, 1994, and in summary inShort Protocols in Molecular Biology, Fifth Edition, Ausubel et al.eds., Current Protocols, 2002.

Antibodies

Antibodies against the marker proteins disclosed herein can be producedusing known methods. These methods of producing antibodies includeimmunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep ormonkey) with the protein. Antibodies may be obtained from immunisedanimals using any of a variety of techniques known in the art, andscreened, preferably using binding of antibody to antigen of interest.Isolation of antibodies and/or antibody-producing cells from an animalmay be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunising a mammal with a protein,an antibody specific for the protein may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, e.g. using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with the protein, or may be one constructed using sequencesobtained from an organism which has been exposed to the antigen ofinterest.

The antibodies may bind or be raised against any biologically relevantstate of the protein. Thus, for example, they can be raised against theunglycosylated form of a protein which exists in the body in aglycosylated form, against a more mature form of a precursor protein,e.g. minus its signal sequence, or against a peptide carrying a relevantepitope of the marker protein.

Antibodies may be polyclonal or monoclonal, and may be multispecific(including bispecific), chimeric or humanised antibodies. Antibodiesaccording to the present invention may be modified in a number of ways.Indeed the term “antibody” should be construed as covering any bindingsubstance having a binding domain with the required specificity. Thus,the invention covers antibody fragments, derivatives, functionalequivalents and homologues of antibodies, including synthetic moleculesand molecules whose shape mimics that of an antibody enabling it to bindan antigen or epitope.

Examples of antibody fragments, capable of binding an antigen or otherbinding partner, are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)₂ fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

Antibody fragments, which recognise specific epitopes, may be generatedby known techniques. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternative, Fab expression libraries may be constructed (Huse, et al.,1989, Science 246: 1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogenous population of antibodies, i.e.the individual antibodies comprising the population are identical apartfrom possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies can be produced by the method firstdescribed by Kohler and Milstein, Nature, 256:495, 1975 or may be madeby recombinant methods, see Cabilly et al, U.S. Pat. No. 4,816,567, orMage and Lamoyi in Monoclonal Antibody Production Techniques andApplications, pages 79-97, Marcel Dekker Inc, New York, 1987.

In the hybridoma method, a mouse or other appropriate host animal isimmunised with the antigen by subcutaneous, intraperitoneal, orintramuscular routes to elicit lymphocytes that produce or are capableof producing antibodies that will specifically bind to the nanoparticlesused for immunisation. Alternatively, lymphocytes may be immunised invitro. Lymphocytes then are fused with myeloma cells using a suitablefusing agent, such as polyethylene glycol, to form a hybridoma cell, seeGoding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986).

The hybridoma cells thus prepared can be seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody producingcells, and are sensitive to a medium such as HAT medium.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the protein.Preferably, the binding specificity is determined by enzyme-linkedimmunoabsorbance assay (ELISA). The monoclonal antibodies of theinvention are those that specifically bind to the protein.

In a preferred embodiment of the invention, the monoclonal antibody willhave an affinity which is greater than micromolar or greater affinity(i.e. an affinity greater than 10⁻⁶ mol) as determined, for example, byScatchard analysis, see Munson & Pollard, Anal. Biochem., 107:220, 1980.

After hybridoma cells are identified that produce neutralisingantibodies of the desired specificity and affinity, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include Dulbecco's ModifiedEagle's Medium or RPM1-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumours in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Nucleic acid encoding the monoclonal antibodies of the invention isreadily isolated and sequenced using procedures well known in the art,e.g. by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of murineantibodies. The hybridoma cells of the invention are a preferred sourceof nucleic acid encoding the antibodies or fragments thereof. Onceisolated, the nucleic acid is ligated into expression or cloningvectors, which are then transfected into host cells, which can becultured so that the monoclonal antibodies are produced in therecombinant host cell culture.

A hybridoma producing a monoclonal antibody according to the presentinvention may be subject to genetic mutation or other changes. It willfurther be understood by those skilled in the art that a monoclonalantibody can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies, humanised antibodies or chimericmolecules which retain the specificity of the original antibody. Suchtechniques may involve introducing DNA encoding the immunoglobulinvariable region, or the complementarity determining regions (CDRs), ofan antibody to the constant regions, or constant regions plus frameworkregions, of a different immunoglobulin. See, for instance, EP 0 184 187A, GB 2 188 638 A or EP 0 239 400 A. Cloning and expression of chimericantibodies are described in EP 0 120 694 A and EP 0 125 023 A.

An antibody against a marker protein described herein will bind to saidprotein. Preferably, said antibody specifically binds said protein. By“specific” is meant that the antibody binds to said protein with anaffinity significantly higher than it displays for other molecules.

The invention will now be described in more detail with reference to thefollowing examples, which are provided for illustrative purposes only,and are not intended to limit the scope of the invention.

All patent and literature references cited herein are herebyincorporated by reference in their entirety.

EXPERIMENTAL

In order to discover novel biomarker candidates for renal allograftrejection, 240 serum samples of kidney transplant recipients from theOxford Transplant Centre were analyzed by expression proteomics. Thesehigh-quality serum samples were depleted of six high-abundant proteinsand analyzed by 2D-electrophoresis. Image analysis and peptide massfingerprinting (PMF) of one half of the serum samples resulted in a setof 29 regulated proteins 20 of which are related to immune andinflammatory response as well as other processes during renal rejectionor renal disease. The remaining 9 proteins have not been described inthe context of renal rejection or renal disease so far. A group of 16proteins of the total number of 29 regulated proteins may serve asindependent indicators for renal allograft rejection. These findingswere confirmed by a validation study run with the second half of theserum samples.

Methods

Renal Rejection Serum Samples

240 serum samples were received from the Oxford Transplant Centre. 120disease serum samples came from 40 kidney transplant recipients withbiopsy proven chronic allograft nephropathy CAN and another 120 controlserum samples came from 40 recipients with a functioning graft withoutevidence of CAN, three different points in time each. Classification ofsamples is shown in table 1 and 2.

TABLE 1 Classification of renal rejection disease serum samples. PointDisease in time Description samples # Time 1 Pre-transplant, close tothe time of 1-1 to 40-1 transplant. Time 2 Before the development ofCAN, but at a time 1-2 to 40-2 when potential mediators of the processmay be circulating (generally at 1 year post transplant). If CAN wasdiagnosed at 1 year, then serum selected at a suitable time point withinthe first year. Time 3 As near as possible to the time of biopsy 1-3 to40-3 diagnosis of CAN

TABLE 2 Classification of renal rejection control serum samples. PointControl in time Description samples # Time 1 Pre-transplant, close tothe time of transplant. 41-1 to 80-1 Time 2 Approximately 1 year posttransplant 41-2 to 80-2 Time 3 Approximately 3 years post transplant41-3 to 80-3

Samples were divided into two groups: study group and validation group.Each group contained samples from 20 disease patients and 20 controlpatients (three different points in time each) which were randomlychosen. For classification of samples see table 3A and 3B.

TABLE 3A Classification of renal rejection serum samples for studygroup. Disease- Disease- Disease- Control- Control- Control- 1 2 3 1 2 31 −1 1 −2 1 −3 41 −1 41 −2 41 −3 5 −1 5 −2 5 −3 42 −1 42 −2 42 −3 8 −1 8−2 8 −3 45 −1 45 −2 45 −3 12 −1 12 −2 12 −3 47 −1 47 −2 47 −3 15 −1 15−2 15 −3 50 −1 50 −2 50 −3 16 −1 16 −2 16 −3 51 −1 51 −2 51 −3 17 −1 17−2 17 −3 53 −1 53 −2 53 −3 18 −1 18 −2 18 −3 54 −1 54 −2 54 −3 19 −1 19−2 19 −3 58 −1 58 −2 58 −3 20 −1 20 −2 20 −3 59 −1 59 −2 59 −3 21 −1 21−2 21 −3 64 −1 64 −2 64 −3 22 −1 22 −2 22 −3 65 −1 65 −2 65 −3 23 −1 23−2 23 −3 68 −1 68 −2 68 −3 24 −1 24 −2 24 −3 69 −1 69 −2 69 −3 26 −1 26−2 26 −3 70 −1 70 −2 70 −3 28 −1 28 −2 28 −3 71 −1 71 −2 71 −3 29 −1 29−2 29 −3 76 −1 76 −2 76 −3 30 −1 30 −2 30 −3 78 −1 78 −2 78 −3 35 −1 35−2 35 −3 79 −1 79 −2 79 −3 39 −1 39 −2 39 −3 80 −1 80 −2 80 −3

TABLE 3B Classification of renal rejection serum samples for validationgroup. Disease- Disease- 1 2 Disease-3 Control-1 Control-2 Control-3 2−1 2 −2 2 −3 43 −1 43 −2 43 −3 3 −1 3 −2 3 −3 44 −1 44 −2 44 −3 4 −1 4−2 4 −3 46 −1 46 −2 46 −3 6 −1 6 −2 6 −3 48 −1 48 −2 48 −3 7 −1 7 −2 7−3 49 −1 49 −2 49 −3 9 −1 9 −2 9 −3 52 −1 52 −2 52 −3 10 −1 10 −2 10 −355 −1 55 −2 55 −3 11 −1 11 −2 11 −3 56 −1 56 −2 56 −3 13 −1 13 −2 13 −357 −1 57 −2 57 −3 14 −1 14 −2 14 −3 60 −1 60 −2 60 −3 25 −1 25 −2 25 −361 −1 61 −2 61 −3 27 −1 27 −2 27 −3 62 −1 62 −2 62 −3 31 −1 31 −2 31 −363 −1 63 −2 63 −3 32 −1 32 −2 32 −3 66 −1 66 −2 66 −3 33 −1 33 −2 33 −367 −1 67 −2 67 −3 34 −1 34 −2 34 −3 72 −1 72 −2 72 −3 36 −1 36 −2 36 −373 −1 73 −2 73 −3 37 −1 37 −2 37 −3 74 −1 74 −2 74 −3 38 −1 38 −2 38 −375 −1 75 −2 75 −3 40 −1 40 −2 40 −3 77 −1 77 −2 77 −3Study Design

Two types of image analysis were performed: (1) time course study ofcontrol and disease patients and (2) control vs. disease patients.2D-gels of samples of the study group were compared as follows:

disease-1 vs. disease-3, control-1 vs. control-3, disease-1 vs.control-1, disease-3 vs. control-3 disease-1 vs. disease-2, disease-2vs. disease-3, control-1 vs. control-2, control-2 vs. control-3.

Again, numbers 1, 2 and 3 refer to the three different points in timesamples were taken. Then the outcome of analytical 2D-gels of thevalidation group was compared with that of the study group to validateregulated protein spots and potential biomarker candidates of the studygroup. For preparative 2D-gels, 6 to 7 serum samples of disease andcontrol samples of the study and the validation group were pooled,respectively.

Proteomics Procedure

The proteomics procedure was done according to standard procedures ofProteome Sciences R&D. In brief, 240 renal rejection serum samples weredepleted of 6 high-abundant proteins by means of the MARS (MultipleAffinity Removal System) column (Agilent). Depleted serum samples (100μg) were analyzed by analytical 2-DE (pH 3-10NL, 10% SDS-PAGE).Silver-stained analytical 2D-gels were quantitatively analyzed byProgenesis software (v2005) including spot detection, matching,background subtraction and normalization. After detection and matchingof protein spots, the spot data was exported to MS Excel and a macrodeveloped in-house was used to calculate the coefficient of variation(CV %), Mann-Whitney Test and regulation factor. Regulated spots wereidentified by the following selection criteria: spots have to be foundin at least 60% of all gels of a group, up/down regulation at least1.5-fold or at most 0.667-fold and significance value p<0.005. In orderto identify the corresponding proteins, regulated spots were picked frompreparative, CBB-stained 2D-gels of depleted disease and control samples(350 μg, 6 to 7 individual samples each) and trypsinated by means of aSpot Handling Workstation (GE Healthcare). Resulting peptides wereanalyzed by MALDI-MS. The proteins were identified by comparing theobtained MALDI-MS-spectra with in-silico digests of proteins fromdatabases (PMF Peptide Mass Fingerprinting).

Results

Identification of Regulated Proteins

Identification of regulated protein spots is shown in table 4 and table5. Histograms of the time course of protein regulations from disease andcontrol samples of point in time 1, 2 and 3 are listed in supplement B.

TABLE 4 Identification of regulated protein spots in studygroup/validation group. Swiss- Expression ratio or trend of Spot ProtEMBL regulation in comparison no Protein name Acc. No. Acc No C1 vs. C3D1 vs. D3 C1 vs. D1 C3 vs. D3 61 Plasminogen P00747 1.76/1.14 + + −precursor 210 Ig μ chain C region P01871 X17115 3.72/2.32 + − − 213 Ig μchain C region P01871 X17115 3.75/2.35 + + − 214 Ig μ chain C regionP01871 X17115 3.12/2.01 + − − 246 C4b-binding M31452; M31452; + + +1.86/0.84 protein alpha BC022312 BC022312 chain precursor 272 HeparinP05546 M12849; + − 1.52/0.81 + cofactor II X03498 precursor 286N-acetylmuramoyl- Q96PD5 AF384856 + − 2.17/0.86 + L-alanine amidaseprecursor 291 Complement P02748 BC020721; + − 1.51/1.50 + component C9K02766 precursor, chain a or b 445 β₂-glycoprotein P02749 X58100;0.46/0.53 + − + I precursor X53595; (Apolipoprotein X57847; H) M62839;S80305; BC020703; BC026283 453 Complement P00736 0.41/0.65 − − − C1Rsubcomponent precursor, heavy chain 461 β₂-glycoprotein P02749 See0.22/0.40 − − + I precursor above (Apolipoprotein H) 466 β₂-glycoproteinP02749 See 0.42/0.59 + − + I precursor above (Apolipoprotein H) 553Pigment P36955 M76979; 0.45/0.96 − − + epithelium- AF400442 derivedfactor precursor 555 Leucine-rich α₂- P02750 AF403428; − + − 4.39/1.40glycoprotein BC034389; precursor BC070198 557 Pigment P36955 See0.51/0.98 − − + epithelium- above derived factor precursor 559 PigmentP36955 See 0.62/0.96 − − + epithelium- above derived factor precursor563 Pigment P36955 See 0.33/0.55 − − + epithelium- above derived factorprecursor 570 Pigment P36955 See 0.32/0.69 − − 2.11/0.91 epithelium-above derived factor precursor 591 Complement P01024 K02765 − +0.43/0.57 + C3 precursor 712 Transthyretin P02766 0.37/0.640.31/0.27 + + precursor, syn: Prealbumin 722 AMBP protein P02760 X042250.16/0.16 + − 4.92/2.89 precursor contains: alpha- 1-microglobulin 728AMBP protein P02760 X04225 0.04/0.04 0.24/0.32 + 7.72/9.79 precursor(α₁- microglobulin) 730 AMBP protein P02760 X04225 0.07/0.64 0.37/0.21 +6.55/4.36 precursor (α₁- microglobulin) 734 AMBP protein P02760 X042250.14/0.16 0.54/0.33 + 3.82/2.02 precursor (α₁- microglobulin) 735 AMBPprotein P02760 X04225 0.23/0.04 − − 3.40/2.50 precursor (α₁-microglobulin) 741 AMBP protein P02760 X04225 0.44/0.57 0.29/0.56 + −precursor (α₁- microglobulin) 742 Adiponectin Q15848 D45371 − + −6.89/4.22 760 Complement P36980 0.20/0.44 − − + factor H-related protein2 precursor 771 Complement P01024 K02765 − 0.43/0.54 + − C3 precursor, αchain 781 Complement P01024 K02765 0.51/0.75 − − − C3 precursor, α-chain781 Complement P01028 0.51/0.75 − − − C4 precursor, acidic or basic C4785 Complement P00746 M84526 0.31/0.56 0.53/0.54 − + factor D precursor786 Complement P00746 M84526 — 0.18/0.21 − + factor D precursor 825Plasma retinol- P02753 X00129; 0.26/0.40 — + + binding protein BC020633precursor 826 Plasma retinol- P02753 See 0.27/0.18 0.33/0.47 + + bindingprotein above precursor 847 β₂-glycoprotein P02749 See 0.39/0.71 — − + Iprecursor above (Apolipoprotein H) no. up-regulated spots  4 − 9 no.down-regulated spots 22 9 − total no. of regulated spots 26 9 9

TABLE 5 Identification of regulated protein spots in study group.Expression ratio in Swiss- comparison Spot Prot/[EMBL] C1 vs. C2 vs. D1vs. D2 vs. no Protein name Acc. No. C2 C3 D2 D3 56 Ceruloplasminprecursor P00450 — — 1.53 (Ferroxidase) [M13699; M13536] 67 Plasminogenprecursor 1.74 — 1.74 — 113 Complement factor B precursor P00751 — —2.02 — 149 α₂-macroglobulin precursor P01023 — — 1.68 — 150α₂-macroglobulin precursor P01023 — — 1.97 — 226 NO PROTEIN FOUND — — —2.59 232 NO PROTEIN FOUND — — — 0.35 238 Inter-α-trypsin inhibitor heavyP19827 — — — 0.31 chain H1 precursor 242 Inter-α-trypsin inhibitor heavyP19827 — — — 0.24 chain H1 precursor 243 Inter-α-trypsin inhibitor heavyP19827 — — 4.09 0.12 chain H1 precursor 249 NO PROTEIN FOUND 2.40 — — —288 NO PROTEIN FOUND — — 0.49 — 291 Complement component C9 P02748 — —0.61 — precursor 352 Hemopexin precursor (β_(1B)- P02790 — 1.57 — —glycoprotein) 381 Hemopexin precursor (β_(1B)- P02790 — 0.60 — —glycoprotein) 418 NO PROTEIN FOUND — 0.38 — — 430 NO PROTEIN FOUND —0.31 — — 442 Kininogen-1 precursor P01042 — 0.64 — — 448 NO PROTEINFOUND — — 0.57 — 463 Complement C1r subcomponent P00736 — — — 0.31precursor 464 Complement C1r subcomponent P00736 — — 20.64  0.04precursor 466 β₂-glycoprotein I precursor P02749 0.31 — — —(Apolipoprotein H) 498 NO PROTEIN FOUND — — 3.20 0.33 510 Hemopexinprecursor (β_(1B)- P02790 — — 2.02 0.65 glycoprotein) 515 Hemopexinprecursor (β_(1B)- P02790 — — — 0.42 glycoprotein) 551 Hemopexinprecursor (β_(1B)- P02790 — — 1.96 — glycoprotein) 557 Pigmentepithelium-derived P36955 0.66 — — — factor precursor 562 Leucine-richα₂-glycoprotein P02750 — — — 2.73 precursor 570 Pigmentepithelium-derived P36955 0.54 — — — factor precursor 575 NO PROTEINFOUND — — 0.27 3.17 599 NO PROTEIN FOUND 5.65 — — — 622 ApolipoproteinA-IV precursor P06727 — — 0.25 2.42 657 Eukar. translation initiationfactor Q14152 — — — 1.68 3 subunit 10 [U58046; U78311] 657 Ceruloplasminprecursor P00450 — — — 1.68 658 Ceruloplasmin precursor P00450 2.97 0.33— — 667 NO PROTEIN FOUND — 0.14 — — 672 NO PROTEIN FOUND — — — 1.88 673130 kDa leucine-rich protein P42704 — 0.08 — — 702 NO PROTEIN FOUND 0.61— — — 711 Complement C4-A precursor P0C0L4 — — 1.94 — 711 ComplementC4-B precursor P0C0L5 — — 1.94 — 722 AMBP protein precursor (α₁- P027600.36 0.43 0.17 6.28 microglobulin) 728 AMBP protein precursor (α₁-P02760 0.26 0.15 0.06 4.30 microglobulin) 730 AMBP protein precursor(α₁- P02760 0.27 0.24 0.32 — microglobulin) 734 AMBP protein precursor(α₁- P02760 0.46 0.31 0.20 2.72 microglobulin) 735 AMBP proteinprecursor (α₁- P02760 0.40 — 0.27 3.19 microglobulin) 771 Complement C3precursor P01024 — — — 0.32 785 Complement factor D precursor P007460.42 — 0.27 — 786 Complement factor D precursor P00746 0.10 — 0.06 — 825Plasma retinol-binding protein P02753 — — 0.27 — precursor 826 Plasmaretinol-binding protein P02753 0.40 — 0.23 — precursor 849 NO PROTEINFOUND — 0.30 — — 854 α_(1B)-glycoprotein precursor P04217 — — — 1.51 no.up-regulated spots 4   1   10    12    no. down-regulated spots 12   12    15    10    total no. of regulated spots 16    13    25    22   

Spot nos. 657, 711 and 781 were assigned to two different proteins(highlighted in grey). ‘Spot number’ is the spot number assigned in thereference gel from Progenesis. ‘Expression ratio’ is the quotient ofmean normalized spot volumes. C₁ vs. C₂ means ratio

$\frac{C_{2}}{C_{1}}$and C₁ vs. C₃ means ratio

$\frac{C_{3}}{C_{1}}.$The ratio is given for statistically significant regulations withsignificance value p<0.005. If the regulation did not meet this value,the trend of regulation is indicated by +/− standing forup-/down-regulation.

Taking all comparisons of the study group in table 4 and 5 together, atotal number of 73 individual spots and a total number of 29 individualproteins were identified as regulated. Expression ratio was between 0.04and 20.64. Coverage of regulated proteins, i.e. percentage of theprimary protein sequence which was covered by trypsinated peptides foundby PMF, was 3%-66% with a mean value of 29%.

Validation of the Results of the Study Group with the Validation Group

An independent set of 120 plasma samples (validation group) has beenanalyzed with the same strategy. For each spot found to be regulated inthe study group (see table 4 and 5), the regulation was compared withthe validation group as presented in table 6. The mean validation ratewas 74%.

TABLE 6 Number of regulated spots in study and validation group andvalidation rate. Validation Validation Comparison Study group group rate[%] C1 vs. C3 26 20 77 C1 vs. C2 16 13 81 C2 vs. C3 13 7 54 Control(total) 55 40 73 D1 vs. D3 10 10 100 D1 vs. D2 25 19 76 D2 vs. D3 22 1255 Disease (total) 57 41 72 C1 vs. D1 4 2 50 C3 vs. D3 9 8 89 Inter(total) 13 10 77Functions and Properties of the Regulated Proteins

Below functions and properties of identified proteins are listedaccording to the Swiss-Prot protein knowledgebase [1].

Inflammatory and Immune-Related Proteins in Alphabetical Order

N-Acetylmuramoyl-L-Alanine Amidase Precursor PGRP2_HUMAN Q96PD5

May play a scavenger role by digesting biologically active peptidoglycan(PGN) into biologically inactive fragments. Has no direct bacteriolyticactivity. Strongly expressed in liver and fetal liver, and secreted intoserum. Expressed to a much lesser extent in transverse colon, lymphnodes, heart, thymus, pancreas, descending colon, stomach and testis.Isoform 2 is not detected in the liver or serum. PGRP2_HUMAN is involvedin the immune response.

C4b-Binding Protein α-Chain Precursor C4BP_HUMAN P04003

Controls the classical pathway of complement activation. It binds as acofactor to C3b/C4b inactivator (C3bINA), which then hydrolyzes thecomplement fragment C4b. It also accelerates the degradation of theC4bC2a complex (C3 convertase) by dissociating the complement fragmentC2a. Alpha chain binds C4b. It interacts also with anticoagulant proteinS and with serum amyloid P component. Found in chylomicrons (largelipoprotein particles) in plasma. C4BP_HUMAN is involved in thecomplement pathway, which is part of the innate immune system.

Complement C1R Subcomponent Precursor C1R_HUMAN P00736

C1r B chain is a serine protease that combines with C1q and C1s to formC1, the first component of the classical pathway of the complementsystem. C1R_HUMAN is a component of the complement pathway. It containsa heavy chain and a light chain.

Complement C3 Precursor CO3_HUMAN P01024

C3 plays a central role in the activation of the complement system. Itsprocessing by C3 convertase is the central reaction in both classicaland alternative complement pathways. After activation C3b can bindcovalently, via its reactive thiolester, to cell surface carbohydratesor immune aggregates. Derived from proteolytic degradation of complementC3, C3a anaphylatoxin is a mediator of local inflammatory process. Itinduces the contraction of smooth muscle, increases vascularpermeability and causes histamine release from mast cells and basophilicleukocytes. CO3_HUMAN is involved in inflammatory response¹. Processingof CO3_HUMAN by the removal of 4 Arg residues forms two chains, beta andalpha, linked by a disulfide bond.

Complement C4 Precursor CO4A_HUMAN, CO4B_HUMAN P01028

C4 plays a central role in the activation of the classical pathway ofthe complement system. It is processed by activated C1 which removesfrom the alpha chain the C4a anaphylatoxin. The remaining alpha chainfragment C4b is the major activation product and is an essential subunitof the C3 convertase (C4b2a) and the C5 convertase (C3bC4b2a) enzymes ofthe classical complement pathway. Derived from proteolytic degradationof complement C4, C4a anaphylatoxin is a mediator of local inflammatoryprocess. It induces the contraction of smooth muscle, increases vascularpermeability and causes histamine release from mast cells and basophilicleukocytes. Human complement component C4 is polymorphic at two loci,C4A and C4B. 13 alleles of C4A and 22 alleles of C4B have been detected.The allele shown here is C4A4. The C4A alleles carry the Rodgers (Rg)while the C4B alleles carry the Chido (Ch) blood group antigens. C4Aallotypes react more rapidly with the amino group of peptide antigenswhile C4B allotypes react more rapidly with the hydroxyl group ofcarbohydrate antigens. CO4_HUMAN is a component of the complementpathway and the inflammatory response.

Complement Component C9 Precursor CO9_HUMAN P02748

CO9_HUMAN is the final component of the complement system to be added inthe assembly of the membrane attack complex. It is able to enter lipidbilayers, forming transmembrane channels. Component of the membraneattack complex which groups the complement plasma glycoproteins C5b, C6,C7, C8 and polymeric C9 on biological membranes. CO9_HUMAN is acomponent of the complement alternative pathway² and the membrane attackcomplex³. Thrombin cleaves CO9_HUMAN to produce C9a and C9b.

Complement Factor B Precursor CFAB_HUMAN P00751

Factor B which is part of the alternate pathway of the complement systemis cleaved by factor D into 2 fragments: Ba and Bb. Bb, a serineprotease, then combines with complement factor 3b to generate the C3 orC5 convertase. It has also been implicated in proliferation anddifferentiation of preactivated B-lymphocytes, rapid spreading ofperipheral blood monocytes, stimulation of lymphocyte blastogenesis andlysis of erythrocytes. Ba inhibits the proliferation of preactivatedB-lymphocytes.

Complement Factor D Precursor CFAD_HUMAN P00746

Factor D cleaves factor B when the latter is complexed with factor C3b,activating the C3bbb complex, which then becomes the C3 convertase ofthe alternate pathway. Its function is homologous to that of C1s in theclassical pathway. CFAD_HUMAN is involved in the complement alternatepathway.

Ig μ Chain C Region MUC_HUMAN P01871

Other Proteins in Alphabetical Order

130 kDa Leucine-Rich Protein LPPRC_HUMAN P42704

Unknown function. Expressed ubiquitously. Expression is highest inheart, skeletal muscle, kidney and liver, intermediate in brain,nonmucosal colon, spleen and placenta, and lowest in small intestine,thymus, lung and peripheral blood leukocytes.

Adiponectin ADIPO_HUMAN Q15848

Important negative regulator in hematopoiesis and immune systems; may beinvolved in ending inflammatory responses through its inhibitoryfunctions. Inhibits endothelial NF-kappa-B signaling through acAMP-dependent pathway. Inhibits TNF-alpha-induced expression ofendothelial adhesion molecules. Involved in the control of fatmetabolism and insulin sensitivity. Synthesized exclusively byadipocytes and secreted into plasma.

AMBP Protein Precursor (α₁-Microglobulin) AMBP_HUMAN P02760

AMBP_HUMAN contains 2 chains, α1-microglobulin and inter-α-trypsininhibitor light chain. α1-microglobulin occurs in many physiologicalfluids including plasma, urine, and cerebrospinal fluid. It appears notonly as a free monomer but also in complexes with IgA and albumin. It isan immunomodulatory protein with a broad spectrum of possible clinicalapplications and seems a promising marker for evaluation of tubularfunction. AMBP might be a valuable complement to serum creatinine levelsin the evaluation of renal function in renal transplant recipients.Expressed by the liver and secreted in plasma.

Apolipoprotein A-IV Precursor APOA4_HUMAN P06727

May have a role in chylomicrons and VLDL secretion and catabolism.Required for efficient activation of lipoprotein lipase by ApoC-II;potent activator of LCAT. Apoa-IV is a major component of HDL andchylomicrons. Synthesized primarily in the intestine and secreted inplasma.

Ceruloplasmin Precursor CERU_HUMAN P00450

Ceruloplasmin is a blue, copper-binding (6-7 atoms per molecule)glycoprotein. It has ferroxidase activity oxidizing iron(II) toiron(III) without releasing radical oxygen species. It is involved iniron transport across the cell membrane.

Complement Factor H-Related Protein 2 Precursor FHR2_HUMAN P36980

Might be involved in complement regulation. Can associate withlipoproteins and may play a role in lipid metabolism. Expressed by theliver and secreted in plasma.

Eukaryotic Translation Initiation Factor 3 Subunit 10 IF3A_HUMAN Q14152

Binds to the 40S ribosome and promotes the binding of methionyl-tRNAiand mRNA.

α_(1B)-Glycoprotein Precursor A1BG_HUMAN P04217

Function not known.

β₂-Glycoprotein I Precursor (Apolipoprotein H) APOH_HUMAN P02749

Binds to various kinds of negatively charged substances such as heparin,phospholipids, and dextran sulfate. May prevent activation of theintrinsic blood coagulation cascade by binding to phospholipids on thesurface of damaged cells. It is expressed by the liver and secreted inplasma.

Hemopexin Precursor (β_(1B)-Glycoprotein) HEMO_HUMAN P02790

Binds heme and transports it to the liver for breakdown and ironrecovery, after which the free hemopexin returns to the circulation.Expressed by the liver and secreted in plasma.

Heparin Cofactor II Precursor HEP2_HUMAN P05546

Thrombin inhibitor activated by the glycosaminoglycans, heparin ordermatan sulfate. In the presence of the latter, HC-II becomes thepredominant thrombin inhibitor in place of antithrombin III (AT-III).Also inhibits chymotrypsin, but in a glycosaminoglycan-independentmanner. Expressed predominantly in liver.

Inter-α-Trypsin Inhibitor Heavy Chain H1 Precursor ITIH1_HUMAN P19827

May act as a carrier of hyaluronan in serum or as a binding proteinbetween hyaluronan and other matrix protein, including those on cellsurfaces in tissues to regulate the localization, synthesis anddegradation of hyaluronan which are essential to cells undergoingbiological processes. Contains a potential peptide which could stimulatea broad spectrum of phagocytotic cells.

Kininogen-1 Precursor KNG1_HUMAN P01042

(1) Kininogens are inhibitors of thiol proteases; (2) HMW-kininogenplays an important role in blood coagulation by helping to positionoptimally prekallikrein and factor XI next to factor XII; (3)HMW-kininogen inhibits the thrombin- and plasmin-induced aggregation ofthrombocytes; (4) the active peptide bradykinin that is released fromHMW-kininogen shows a variety of physiological effects: (4A) influencein smooth muscle contraction, (4B) induction of hypotension, (4C)natriuresis and diuresis, (4D) decrease in blood glucose level, (4E) itis a mediator of inflammation and causes (4E1) increase in vascularpermeability, (4E2) stimulation of nociceptors (4E3) release of othermediators of inflammation (e.g. prostaglandins), (4F) it has acardioprotective effect (directly via bradykinin action, indirectly viaendothelium-derived relaxing factor action); (5) LMW-kininogen inhibitsthe aggregation of thrombocytes; (6) LMW-kininogen is in contrast toHMW-kininogen not involved in blood clotting. It is a secreted protein

Leucine-Rich α₂-Glycoprotein Precursor A2GL_HUMAN P02750

It is a protein secreted in plasma.

α2-Macroglobulin Precursor A2MG_HUMAN P01023

Is able to inhibit all four classes of proteinases by a unique‘trapping’ mechanism. This protein has a peptide stretch, called the‘bait region’ which contains specific cleavage sites for differentproteinases. When a proteinase cleaves the bait region, a conformationalchange is induced in the protein which traps the proteinase. Theentrapped enzyme remains active against low molecular weight substrates(activity against high molecular weight substrates is greatly reduced).

Following cleavage in the bait region a thioester bond is hydrolyzed andmediates the covalent binding of the protein to the proteinase

Pigment epithelium-derived factor precursor PEDF_HUMAN P36955

Neurotrophic protein; induces extensive neuronal differentiation inretinoblastoma cells. Potent inhibitor of angiogenesis. As it does notundergo the S (stressed) to R (relaxed) conformational transitioncharacteristic of active serpins, it exhibits no serine proteaseinhibitory activity. It is a secreted protein and is found in retinalpigment epithelial cells and blood plasma.

Plasma Retinol-Binding Protein Precursor RETBP_HUMAN P02753

Delivers retinol from the liver stores to the peripheral tissues. Inplasma, the RBP-retinol complex interacts with transthyretin, thisprevents its loss by filtration through the kidney glomeruli. It is asecreted protein.

Plasminogen Precursor PLMN_HUMAN P00747

Plasmin, formed by conversion from plasminogen, dissolves the fibrin ofblood clots and acts as a proteolytic factor in a variety of otherprocesses including embryonic development, tissue remodeling, tumorinvasion, and inflammation; inovulation it weakens the walls of theGraafian follicle. It activates the urokinase-type plasminogenactivator, collagenases and several complement zymogens, such as C1 andC5. It cleaves fibrin, fibronectin, thrombospondin, laminin and vonWillebrand factor. Its role in tissue remodeling and tumor invasion maybe modulated by CSPG4. It is a secreted protein and is present in plasmaand many other extracellular fluids. It is synthesized in the kidney.

Transthyretin Precursor TTHY_HUMAN (syn Prealbumin) P02766

Thyroid hormone-binding protein. Probably transports thyroxine from thebloodstream to the brain. It is a secreted protein. Most abundant in thechoroid plexus and also present in the liver. About 40% of plasmatransthyretin circulates in a tight protein-protein complex with theplasma retinol-binding protein (RBP). The formation of the complex withRBP stabilizes the binding of retinol to RBP and decreases theglomerular filtration and renal catabolism of the relatively small RBPmolecule.

Conclusion

In this study a set of proteins was identified as biomarker candidates.Results from the study group and the validation group showed anexcellent correlation in protein regulation which proves the highquality of the serum sample set and the proteomics procedure.

Time Course of Protein Regulations

Histograms of the time course of protein regulations from disease andcontrol samples of point in time 1, 2 and 3 show three strikingregulations, namely Ig μ chain C region (spot no. 210, 213 and 214),pigment epithelium-derived factor precursor (spot no. 570) and AMBPprotein precursor (α₁-microglobulin) (spot no. 722, 728, 730 and 735).Ig μ level steadily increases in healthy recipients whereas it increasesfrom point in time 1 to 2 and decreases again from point in time 2 to 3in rejecting recipients. Pigment epithelium-derived factor precursor andα₁-microglobulin levels steadily decrease in healthy recipients whereasthey decrease from point in time 1 to 2 and increase again from point intime 2 to 3 in rejecting recipients. The same expression profile isobserved with spots no. 445, 461, 562, 657, 734, 785, 825, 826, 854 and862 (see table 4 and 5).

Biomarker Candidates

From the individual comparisons and the time course regulations 16biomarker candidates could be identified (Swiss-Prot/EMBLprotein/nucleic acid database accession numbers are given):

N-acetylmuramoyl-L-alanine amidase precursor Q96PD5/AF384856

Adiponectin Q15848/D45371

AMBP protein precursor (α₁-microglobulin) P02760/X04225

C4b-binding protein α-chain precursor P04003/M31452; BC022312

Ceruloplasmin precursor P00450/M13699; M13536

Complement C3 precursor P01024/K02765

Complement component C9 precursor P02748/BC020721; K02766

Complement factor D precursor P00746/M84526

α_(1B)-glycoprotein P04217/AF414429; BC035719

β₂-glycoprotein I precursor P02749/X58100; X53595; X57847; M62839;S80305; BC020703; BC026283

Heparin cofactor II precursor P05546/M12849; X03498

Ig μ Chain C region protein P01871/X17115

Leucine-rich α₂-glycoprotein precursor P02750/AF403428; BC034389;BC070198

Pigment epithelium-derived factor precursor P36955/M76979; AF400442

Plasma retinol-binding protein precursor P02753/X00129; BC020633

Translation initiation factor 3 subunit 10 Q14152/U58046; U78311

Conclusion

240 serum samples of kidney transplant recipients were submitted to 2DEproteomic procedure in order to identify novel biomarker candidates forearly detection of renal rejection. The clear correlation of the resultsof the study group and the validation group shows the robustness andreproducibility of our proteomics procedure, especially serum samplepreparation by chromatographic immunoaffinity depletion of six highabundant proteins which is commercially available only since 2003, aswell as the high quality of the serum sample set. 29 proteins were foundregulated between the three selected conditions of renal transplantrecipients, 20 of which are related to inflammatory and immune responseduring renal rejection or renal disease. As a result, 16 proteins couldbe identified as novel biomarker candidates.

Statistical Analysis of the 2D Gel Data with SIMCA-P.

Data Pre-Processing

The normalised and background-corrected spot data were exported fromProGenesis to Excel and automatically arranged into a format suitable byan in house-developed programme for further analysis. For eachcomparison of control and treated data, the number of spots in eachgroup, the mean value, the coefficient of variation and two-tailedStudent's t-test and Mann-Whitney Test computed by StatistiXL (Version1.5) were automatically generated. In parallel, the data were importedinto SIMCA-P software (version 11; Umetrics) and the spot data assessedto determine whether any spots required pre-processing prior to furtheranalysis. For the spot data (variables), two different scaling methodswere evaluated; Unit variance (UV) and Pareto (Par). UV is the defaultscaling method and Pareto scaling is in between no scaling and UVscaling and gives the variable a variance equal to its standarddeviation instead of unit variance. Three different transformationmethods were evaluated; no transformation, automatic transformation andLog₁₀ transformation. By default, there is no transformation, theautomatic transformation is performed by the software which checks ifvariables need a log transformation and if so it applies it. The Log₁₀transformation is applied to spots that are positively skewed (>0.7) togive distributions closer to normal. The optimal goodness-of-fit (R²)and cumulative goodness-of-prediction (Q²[cum]) parameters were obtainedwith the combination of the UV scaling method with an automatictransformation for the control (C gels) and disease gels (C gels) asshown in Table 7.

TABLE 7 Scaling and transformation methods tested for the variables.number of Model component Transformation Scaling R2X R2Y Q2(cum) PCAwith C gels 4 no transformation UV 0.354 x 0.0682 PCA with C gels 3 notransformation Pareto 0.322 X 0.0456 PCA with C gels 4 Automatic UV0.398 X 0.0846 PCA with C gels 3 Log10 UV 0.336 X 0.0923 PCA with C-2gels 5 Automatic UV 0.439 X 0.0838 PCA with D gels 3 no transformationUV 0.303 X 0.08 PCA with D gels 3 no transformation Pareto 0.339 X0.0705 PCA with D gels 4 Automatic UV 0.41 X 0.127 PCA with D gels 4Automatic Pareto 0.41 X 0.0951 PCA with D gels 4 Log10 Pareto 0.402 X0.0852

There were two kinds of analysis performed. Initially, an unsupervisedmethod called principal components analysis (PCA) was applied to allexperimental groups. Then secondly, a supervised method called partialleast squares—discriminant analysis (PLS-DA) was applied to the fourdata sets comparing the Control −1 versus Control −3; Disease −1 versusDisease −3; Control −1 versus Disease −1 and Control −3 versus Disease−3 to find out whether any differences could be found to distinguishbetween sample groups.

PCA Analysis of the Control Data Set

Principal component Analysis (PCA) is a multivariate projection methoddesigned to extract and display the systematic variation in the datamatrix (gels and spot volumes in our case). With PCA analysis, a numberof diagnostic and interpretation tools are available.

A four component model was auto-fitted to the control data set. Themodel parameters were as follows: goodness-of-fit (R²)=0.398 and thecumulative goodness-of-prediction (Q²[cum])=0.0846. FIG. 8 shows theobservations scores of the model.

We can observe in the FIG. 8 that two gels are located outside theellipse and could be considered as strong outliers (C45_(—)1 andC46_(—)3). We removed these two gels and a new model with fivecomponents was fitted (see Table 7). The model parameters were asfollows: R²=0.439 and Q²[cum]=0.0838. FIG. 9 shows the observationsscores of the model.

Although the goodness-of-fit (R²) parameter indicates that 44% of thetotal variance in the data are explained, the low value for the Q²suggests that the model is not very robust. As shown in FIG. 9, it isclear that the model does not display clusters between the gels in thegroup. A typical clustering model may display two groups, onecorresponding to the control_(—)1 gels and the second to thecontrol_(—)3 gels.

PCA Analysis of the Disease Data Set

A four component model was auto-fitted to the control data set. Themodel parameters were as follows: goodness-of-fit (R²)=0.41 and thecumulative goodness-of-prediction (Q²[cum])=0.127. FIG. 10 shows theobservations of the model.

Although the goodness-of-fit (R²) parameter indicates that 41% of thetotal variance in the data are explained, the low value for the Q²suggests that the model is not very robust. As shown in FIG. 10, we donot observe outliers, nor does the model display clusters between thegels in the group.

For the two models, we observe low values for R² and near-zero valuesfor Q² leading us to conclude that these models are not well explainedor predicted. Even if low values are already observed for 2D gel data,especially related to human sample analysis, potential explanations arethe samples themselves (high biological heterogeneity) and/or sampletreatments (freeze-thaw cycles, depletion, 2D gel electrophoresis).

PLS-DA of the Control Data Set

The Partial Least Squares—Discriminant Analysis (PLS-DA) is a method forrelating two data matrices, X (gels) and Y (spot volume), to each otherby a linear multivariate model. This type of analysis is appropriate todetermine whether any differences could be found to discriminate betweenthe control_(—)1 group (blood taken during transplantation) andcontrol_(—)3 group (blood taken when the transplantation has beenvalidated).

A two component model was auto-fitted to the control gels data set. Theparameters were as follows: R²X=0.185; R²Y=0.898 and Q²=0.431. Theadditional parameter R²Y described the goodness-of-fit to the Y-datacontaining the class of observations. The observations of the scores inthe two PLS components of the model are displayed in FIG. 11. The valueof Q² close to 0.5 could be considered as good. As seen in FIG. 11, theseparation of the groups appears excellent.

The loading plots of this PLS model are displayed in FIG. 12. In lightertype are indicated the number of spots selected through the univariateapproach. The plots at the extremes of the horizontal axis describe thedifferences between the groups. We can observe in FIG. 12 that thelighter type numbers are located at the extremes of the axis whichreinforces the fact that these spots may reflect a significantdiscrimination between the two groups compared. This observation shows agood correlation between the univariate and multivariate approaches aswell.

PLS-DA of the Disease Data Set

We applied this type of analysis to determine whether any differencescould be found to discriminate between the disease_(—)1 group (bloodtaken during transplantation) and disease_(—)3 group (blood taken whenthe transplantation has been rejected).

A two component model was auto-fitted to the control gels data set. Theparameters were as follows: R²X=0.143; R²Y=0.902 and Q²=0.301. Theobservations of the scores in the two PLS components of the model aredisplayed in FIG. 13. As seen in FIG. 13, the separation of the groupsappears excellent.

The loading plots of this PLS model are displayed in FIG. 14. In lightertype are indicated the number of spots selected through the 2D gelapproach. We can observe in FIG. 14 that the lighter type numbers arelocated at the extremes of the axis which reinforces the fact that thesespots may reflect a significant discrimination between the two groupscompared. This observation shows a good overlap between the univariateand multivariate approaches as well.

Analysis of the Data Set Containing Control_(—)1 and Disease_(—)1(“Sample_(—)1”) Groups

PCA Analysis of the Sample_(—)1 Data Set

A six component model was auto-fitted to the control data set. The modelparameters were as follows: goodness-of-fit (R²)=0.481 and thecumulative goodness-of-prediction (Q²[cum])=0.0519. FIG. 15 shows theobservations plots of the model.

Although the goodness-of-fit (R²) parameter indicates that 48% of thetotal variance in the data are explained, the low value for the Q²suggests that the model is not very robust. As shown in FIG. 15, we donot observe strong outliers, nor does the model display clusters betweenthe gels in the group.

PLS-DA Analysis of the Sample_(—)1 Data Set

A two component model was auto-fitted to the data set. The parameterswere as follows: R²X=0.199; R²Y=0.828 and Q²=0.148. The observations ofthe scores in the two PLS components of the model are displayed in FIG.16. As seen in FIG. 16, the separation of the groups appears excellent.

The loading plots of this PLS model are displayed in FIG. 17. In lightertype are indicated the number of spots selected through the 2D gelapproach. We can observe in FIG. 17 that the lighter type numbers arelocated at the extremes of the axis which reinforces the fact that thesespots may reflect a significant discrimination between the two groupscompared. This observation shows a good overlap between the statisticaland 2D gel electrophoresis approaches as well.

Analysis of the Data Set Containing Control_(—)3 and Disease_(—)3(“Sample_(—)3”) Groups

PCA Analysis of the Sample_(—)3 Data Set

During a preliminary analysis with all gels from sample_(—)3 set, weobserved a strong outlier (gel D12_(—)3, data not shown). Consequently,we removed the gel from the analysis and performed a new one. A sixcomponent model was auto-fitted to the sample_(—)3 data set. The modelparameters are R²=0.488 and Q²=0.0757. The observation scores for thetwo PCA components of this model are displayed in FIG. 18.

The low values for R² and Q² suggest that the model is not particularlyrobust. It is clear from the examining the positions of the gels in thefirst two components that there is no pattern of clustering betweenobservations in this data set.

PLS-DA Analysis of the Sample_(—)3 Data Set

A four component model was auto-fitted to the data set. The parameterswere as follows: R²X=0.302; R²Y=0.992 and Q²=0.725. Generally, a Q²>0.7is regarded as very good. The observations of the scores in the two PLScomponents of the model are displayed in FIG. 12. As seen in FIG. 19,the separation of the groups appears excellent.

The loading plots of this PLS model are displayed in FIG. 20. In lightertype are indicated the number of spots selected through the 2D gelapproach. We can observe in FIG. 20 that the lighter type numbers arelocated at the extremes of the axis which reinforces the fact that thesespots may reflect a significant discrimination between the two groupscompared. This observation shows a good overlap between the statisticaland 2D gel electrophoresis approaches as well.

The invention claimed is:
 1. A method for the diagnosis and/or prognosisof renal damage in a human patient, said renal damage being caused bychronic transplant rejection following renal transplantation, the methodcomprising (i) detecting the presence or amount of an autoantibodyagainst at least one marker protein in a sample from said patient, saidautoantibody being at least one of an IgG autoantibody and an IgMautoantibody, said sample being selected from the group consisting of arenal tissue sample, a blood sample, a serum sample, a plasma sample anda urine sample; (ii) comparing the detected autoantibody to a controlsample, wherein the presence or altered amount of said at least onemarker protein in said patient sample, as represented by said detectedpresence or amount of the autoantibody, in comparison to saidautoantibody level in said control sample, is indicative of said renaldamage in said patient, and wherein said at least one marker protein isselected from the group consisting of Ig μ chain C region protein;C4b-binding protein α-chain precursor; heparin cofactor II precursor;N-acetylmuramoyl-L-alanine amidase precursor; complement component C9precursor; β2-glycoprotein I precursor; leucine-rich α₂-glycoproteinprecursor; complement C3 precursor; AMBP protein precursor(α-1-microglobulin); adiponectin; complement factor D precursor;ceruloplasmin precursor (ferroxidase); pigment epithelium-derived factorprecursor; translation initiation factor 3 subunit 10; plasmaretinol-binding protein precursor; α_(1B)-glycoprotein.
 2. The method ofclaim 1, wherein the renal damage is caused by chronic allograftnephropathy.
 3. The method of claim 2, wherein said marker protein isN-acetylmuramoyl-L-alanine amidase precursor.
 4. The method of claim 1,wherein the method comprises the steps of: (a) contacting said samplefrom a patient with a solid support having immobilised thereon a bindingagent having binding sites which are capable of specifically binding tosaid autoantibody under conditions in which said autoantibody binds tothe binding agent; and, (b) determining the presence or amount of saidautoantibody bound to the binding agent.
 5. The method of claim 4,wherein step (b) comprises (i) contacting the solid support with adeveloping agent which is effective to bind to occupied binding sites,unoccupied binding sites or said autoantibody, the developing agentcomprising a label and (ii) detecting the label to obtain a valuerepresentative of the presence or amount of said autoantibody in saidsample.
 6. The method of claim 1, wherein the method comprises detectingsaid autoantibody by Western blot, enzyme-linked immunosorbent assay(ELISA), protein microarray or bead suspension array using purifiedproteins.
 7. The method of claim 6, wherein the protein is immobilisedon a solid support.
 8. The method of claim 5, wherein the label is aradioactive label, a fluorophor, a phosphor, a laser dye, a chromogenicdye, a macromolecular colloidal particle, a latex bead which iscoloured, magnetic or paramagnetic, an enzyme which catalyses a reactionproducing a detectable result or the label is a tag.
 9. The method ofclaim 4, which comprises detecting the presence or amount of a pluralityof said autoantibodies associated with chronic transplant rejection in asingle sample.
 10. The method of claim 9, wherein the method employs aplurality of binding agents which are immobilised at predefinedlocations on the solid support.